HER2-enriched Breast Cancer Research Articles

Disparities in minority patients with breast cancer under 40 years of age: A single center experience.

e18553 Background: Current guidelines recommend screening for breast cancer (BC) starting at age 40. However, BC in women under 40 years is increasing in incidence. Pathogenic germline mutations are found to be the driving force in nearly 17% of those diagnosed under 45 years and 50% of those under 30 years. Limited data is available on the prevalence of pathogenic mutations in minority patients. We conducted a retrospective analysis of young breast cancer patients from low socioeconomic backgrounds between 2010-2022 at a single center in Central Texas. Methods: Patients were selected based on a diagnosis of BC under 40 years old and living at or below 200% of federal poverty level as noted through their insurance status of Medicaid and Medical Assistance Program of Travis County. Data regarding age of diagnosis, demographics, clinical presentation, staging, germline mutations and outcomes were extracted from the medical record. Descriptive statistics were used to analyze the dataset. Results: A total of 125 patients were identified for the study. Median age of the cohort was 35 years old (range: 19-39 years). Demographics included 61 Hispanic patients (48.8%), 40 White patients (32.0%), 16 Black patients (12.8%) and < 2% for each other group. Analysis of germline mutations was available for 86 patients. High risk mutations were identified in 28 patients, including 13 Caucasian women, 10 Hispanic women, and three Black women. BRCA mutations were the most common mutation (75%). Other mutations included CHEK2 (7.2%), MUTYH (7.2%), ATM (3.6%), PIK3CA (3.6%), PTEN (3.6%), RAD51C (3.6%) and RAD51D (3.6%). Variants of unknown significance were found in 18 patients, including 11 Hispanic women, four Black women and two White women. Forty patients had BC independent of mutations. Staging on initial diagnosis was available for 113 patients, 40.7% of which were locally advanced (28.3% in Stage III disease and 12.4% with metastatic disease). Advanced disease was higher in Hispanic patients (22/61) and Black patients (10/16) compared to White patients (10/40). Breast cancer subtypes included 22.8% with triple negative BC, 8.1% with HER2-enriched BC, 27.9% with hormone receptor (HR) positive and HER2 negative BC and 14.0% with both HR and HER2 positive BC. At the time of data review, only 30 patients were documented to have died, of which 22 died within five years of diagnosis. Five year death rate was 26.2% for Hispanic patients, 18.8% for Black patients and 7.5% for White patients in the study cohort. Conclusions: This study shows disparities among young Hispanic and Black patients with BC, particularly in the initial staging of BC and 5-year death rate. A large number of VUS were seen in minorities, suggesting little is known about the pathogenic nature of these mutations. Future prospective studies should consider investigating interplay between race and outcomes of those diagnosed with BC under 40 years old.

PREDICTING RESPONSE TO NEOADJUVANT CHEMOTHERAPY IN OPERABLE BREAST CANCER: ROLE OF ER, PR, HER2 AND KI-67 INDEX

Background: Neoadjuvant (anterior or pre-operative) chemotherapy for operable breast cancer helps in down-staging of the tumor and increases the rate of breast conservation surgery. Factors that identify individuals who benet from chemotherapy have not been clearly established. This study was conducted to assess the role of estrogen receptor, progesterone receptor, HER2 and Ki-67 index in predicting response to a standard anthracycline and taxane-based chemotherapy. Methods: The study included 62 women with clinical tumor stage 1-3 and nodal stage 0 or 1. All women received preoperative anthracycline and taxane-based chemotherapy followed by surgery. Clinical and pathological response was assessed using World Health Organization (UICC) and Chevallier's criteria respectively. Results: Clinical and pathological complete response rates were 40.3% and 17.7% respectively. The pathological complete response was highest in HER2-enriched patients (45.45%) while basal-like and luminal B subtypes accounted for 27.27% each. Among those that achieved pathological complete response, 90% had Ki-67 >20, 81.8% had grade 3, 72.7% had ER negative status, 81.8% had PR negative status, 63.6% had HER2 positive status. However, there was no statistically signicant association between preoperative clinical tumor size(p=0.98), ER(p=0.053), PR(p=0.17), HER2(p=0.17), Ki-67 index(p=0.26) and pathological complete response. Conclusion: Ahigh pathological complete response was observed in tumors with high grade, high proliferative activity (high Ki-67), ER/PR negative status, HER2 over-expressing tumors and aggressive molecular subtypes such as basal-like and HER2-enriched breast cancer. However, none of these pre-chemotherapy factors showed statistically signicant association with pathological complete response.

Nuclear and Cytoplasmic hTERT, Tumor-Infiltrating Lymphocytes, and Telomere Elongation Leukocytes Are Independent Factors in the Response to Neoadjuvant Treatment in HER2-Enriched Breast Cancer

HER2-enriched tumors are responsible for 20% of breast tumors and have high rates of immune infiltrates in the tumor stroma that respond favorably to neoadjuvant chemotherapy. In the context of tumors, telomeres control cell death and prevent tumor cells from replicating discontinuously, leading to their immortalization. This study aimed to evaluate the presence of tumor-infiltrating lymphocytes, hTERT expression, hTERT promoter mutation, and leukocyte telomere length in HER2-enriched breast tumors. A total of 103 cases were evaluated, 19 with pathologic complete response. The TILs percentage was above ≥10 in 44 cases (43%) and significantly present in patients ≥50 years of age. hTERT staining positivity was mostly nuclear, significantly present in the non-pCR group, and associated with a lower survival rate. Leukocyte telomeres were elongated for HER2-enriched tumors, and in multivariate analysis, shortening was associated with an increased risk of death. Overall, our results show that the nuclear and cytoplasmic presence of hTERT may indicate a worse prognosis and that leukocyte telomere elongation is a protective factor.

Abstract 1721: Dual targeting of JAK2 and TrkA inhibits breast cancer cell stemness, enhances apoptosis, and delays tumor growth of triple-negative and HER2-enriched breast cancers

Abstract Despite the current standard of care, metastatic triple-negative breast cancer (TNBC) and HER2-enriched breast cancer remain difficult to treat due to limited actionable targets, poor response to treatment, and intrinsic/acquired therapeutic resistance. Thus, more effective therapies are needed to reduce the high mortality rates associated with metastatic TNBC and HER2-enriched breast cancer. The Janus Kinase 2 (JAK2)-Signal Transducer and Activator of Transcription 3 (STAT3) pathway is dysregulated in multiple solid tumors, including breast cancer, and is being investigated as a therapeutic target. Similarly, Tropomyosin receptor tyrosine kinase A (TrkA) is an emerging target in metastatic breast cancers. The prevalence of aberrant JAK2 or TrkA signaling led to the development and FDA approval of orally active, selective inhibitors for JAK2 (pacritinib) and TrkA (entrectinib, larotrectinib). However, breast cancer patients receiving either JAK2 or TrkA inhibitors showed suboptimal clinical response, suggesting that oncogenic signaling events are sustained in the absence of either JAK2 or TrkA activity. In support of this paradigm, we previously reported a novel signaling crosstalk between the JAK2-STAT3 and TrkA pathways (Cancers, 10:2340-2360, 2021). We found that JAK2 and TrkA are preferentially co-activated in TNBC and HER2-enriched breast cancer, and both kinases converge to phosphorylate STAT3 to drive gene transcription and enhance breast cancer cell stemness. The observed significant functional crosstalk between JAK2 and TrkA pathways suggest that co-inhibition of these pathways would greatly benefit breast cancer patients with co-activation of these pathways. In this study, we aimed to determine the therapeutic utility of dual targeting JAK2 and TrkA in TNBC and HER2-enriched breast cancer using FDA-approved selective inhibitors. Our data showed that JAK2 and TrkA inhibitors synergize to reduce breast cancer cell viability in vitro. Our study further revealed that co-inhibition of JAK2 and TrkA potently inhibits breast cancer cell stemness since co-inhibition of these kinases significantly reduces the CD44high/CD24low cell population, mammosphere formation, and ALDH activity to a greater extent than vehicle or monotherapies. We also found that co-inhibition of JAK2 and TrkA more effectively induces apoptosis of breast cancer cells, as indicated by Annexin V flow cytometry, Western blot analysis for cleaved PARP, and Caspase 3/7 activity. Importantly, our in vivo studies showed that co-inhibition of JAK2 and TrkA significantly delays mammary tumor growth using cell line-derived and patient-derived xenograft breast cancer mouse models. Taken together, our data illustrates the therapeutic utility of dual targeting JAK2 and TrkA in metastatic TNBC and HER2-enriched breast cancers. Citation Format: Angelina T. Regua, Mariana K. Najjar, Grace L. Wong, Sara G. Manore, Austin Arrigo, Hui-Wen Lo. Dual targeting of JAK2 and TrkA inhibits breast cancer cell stemness, enhances apoptosis, and delays tumor growth of triple-negative and HER2-enriched breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1721.

Abstract 3769: Intracellular miR-1290 promotes breast cancer stemness in HER2-enriched and triple-negative breast cancer

Abstract HER2-enriched breast cancer and triple-negative breast cancer (TNBC) have the highest propensity to metastasize to the brain; patients with breast cancer brain metastasis (BCBM) survive only 6-18 months after diagnosis. Mechanisms that drive brain metastasis remain unclear, contributing to limited effective treatments and poor prognoses for HER2-enriched breast cancer and TNBC patients. Our lab recently reported that breast cancer with truncated glioma-associated oncogene homolog 1, a BCBM-promoting transcription factor, secretes high levels of extracellular vesicle (EV)-derived miR-1290 (Cancer Letters 540:215726, 2022). EV-miR-1290 activates astrocytes in the brain microenvironment. Using mammosphere assays, which enrich breast cancer stem cells (BCSCs), we found conditioned media from miR-1290-activated astrocytes promotes mammosphere formation of breast cancer cells. Furthermore, miR-1290-activated astrocytes secrete high levels of ciliary neurotrophic factor (CNTF) to promote the progression of brain metastases through the novel EV-miR-1290-FOXA2-CNTF signaling axis. However, it remains unknown whether intratumoral miR-1290 promotes BCSCs and BCBM. To help fill this knowledge gap, we first examined whether a miR-1290 mimic enhanced expression of known BCSC markers in SKBR3, HER2-enriched breast cancer, and CN34, TNBC, cells. Our results showed that overexpression of miR-1290 significantly increased the expression of stemness genes, CD44, Nanog, and OCT4, in both cell lines. Furthermore, we determined whether miR-1290 promotes formation of mammospheres that are enriched with BCSCs, and the results indicated that ectopic miR-1290 expression significantly enriched BCSCs in SKBR3 and CN34 cells. Conversely, inhibition of miR-1290 suppressed mammosphere-forming ability of SKBRM and CN34-BRM cells, two brain metastatic breast cancer cell lines derived from SKBR3 and CN34 cells, respectively. We further observed that miR-1290 overexpression in SKBR3 cells significantly increased the percentage of CD44+/CD24− cells, indicative of the BCSCs. Analysis of Gene Expression Omnibus breast cancer patient datasets revealed that miR-1290 expression is significantly increased in HER2-positive and basal subtypes of breast cancer patient tumors. Using the publicly available miR-1290 gene signature, we performed Gene Set Enrichment Analysis and found that high miR-1290 gene activation signature is positively enriched with multiple pathway gene signatures that are known to be upregulated in breast cancer, such as EGFR, MAPK, PI3K, and STAT3 pathways. Furthermore, the miR-1290 gene signature is upregulated in HER2-positive breast cancer and TNBC tumors, and is correlated with worse metastasis-free survival (MFS) and brain-MFS in breast cancer patients. In summary, our study suggests an important role for intratumoral miR-1290 in BCSCs and BCBM. Citation Format: Grace L. Wong, Mariana Najjar, Yoshua Esquenazi, Nitin Tandon, Angelina T. Regua, Hui-Wen Lo. Intracellular miR-1290 promotes breast cancer stemness in HER2-enriched and triple-negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3769.

Predictors of nodal metastases in early stage HER2+ breast cancer: Deciding on treatment approach with neoadjuvant chemotherapy vs. upfront surgery

BackgroundThe purpose of this study is to evaluate preoperative predictors of nodal metastases in patients with early-stage, HER2-positive (HER2+) breast cancer. MethodsThe SEER Database was queried to identify women with a first diagnosis of stage I-II (T1-T2) HER2-positive breast cancer treated with upfront surgery in 2018. Multivariable logistic regression was used to identify clinical characteristics independently associated with nodal involvement. ResultsOverall, 3333 women with stage I-II HER2+ breast cancer met inclusion criteria and were included in the study. The median age at diagnosis was 59 years (IQR, 51–69 years). Most patients underwent breast-conserving surgery (60.9%), with a median of 3 (IQR 2–4) axillary lymph nodes removed. On final pathology, 762 (22.9%) of T1-T2 HER2+ patients were node positive; 2.7% pN0[i+], 3.7% pN1mi, 15.1% pN1, and 1.4% pN2. Women less than 40 years and those between 40 and 49 years showed the highest proportion of axillary lymph node metastasis, in 33.7% and 30.7% respectively, and declining with age (p < 0.001). Patients with triple-positive breast cancer had the highest rates of nodal involvement (24.8%), compared to 20.7% ER+/PR-/HER2+ and 19.6% of HER2-enriched patients (p = 0.006). On adjusted analysis, age, biologic subtype, tumour size, and type of surgery remained independent predictors of nodal involvement. On subgroup analysis, women under age 50 with T1c HER2-enriched or triple-positive breast cancer had a 33% and 35% incidence of nodal involvement, which declined with age. ConclusionsThe likelihood of pathologic nodal involvement in early-stage HER2+ breast cancer is contingent on age, ER/PR status, and tumour size.

The use of neoadjuvant systemic therapies in breast cancer in Australia and New Zealand: Breast Surgeons of Australia and New Zealand quality audit.

Breast surgeons must maintain contemporary knowledge regarding appropriate referral for neoadjuvant chemotherapy (NACT) in breast cancer (BC) patients. To date, the greatest benefit is seen in stage II-III HER2-enriched and triple negative breast cancers (TNBC). This study is the first audit of use of NACT in Australia and New Zealand to stratify data by BC biological subtype. Prospective data from 116,745 patients between 2010 and 2019 was provided by the Breast Surgeons of Australia and New Zealand (BreastSurgANZ) Quality Audit (BQA) of Breast Cancer Care. Annual rates of NACT use were determined and change across time analysed with fractional regression. Data from 2018 to 2019 were combined and stratified by biological subtype (LumA, LumB HER2-neg, LumB HER2-pos, HER2 enriched, TNBC, Other basal-like), and age (<50, 51-74, and ≥75 years) and compared using negative binomial regression. The use of NACT increased annually (OR 1.26, P< 0.001), and the use of additional adjuvant chemotherapy (ACT) decreased (OR 0.78, P< 0.001). A significantly greater use of NACT was noted in patients with TNBC and HER2+ BC, and in all patients aged <50 years compared with older ages (P< 0.001), regardless of biological subtype. Increased uptake of NACT and decreased use of additional ACT is in keeping with progressive change in practice in response to contemporary evidence. Expansion of BQA data fields related to use of NACT, and detailed audit of NACT rates in Stage II-III TNBC and HER2 enriched BC will allow accurate determination of quality of practice in ANZ.

Opposite and dynamic regulation of the interferon response in metastatic and non-metastatic breast cancer

BackgroundTo our current understanding, solid tumors depend on suppressed local immune reactions, often elicited by the interaction between tumor cells and tumor microenvironment (TME) components. Despite an improved understanding of anti-cancer immune responses in the TME, it is still unclear how immuno-suppressive TME are formed and how some cancer cells survive and metastasize.MethodsTo identify the major adaptations that cancer cells undergo during tumor development and progression, we compared the transcriptome and proteome from metastatic 66cl4 and non-metastatic 67NR cell lines in culture versus their corresponding mouse mammary primary tumors. Using confocal microscopy, RT-qPCR, flow cytometry and western blotting, we studied the signaling pathway and the mechanisms involved. In addition, we used public gene expression data from human breast cancer biopsies to evaluate the correlation between gene expression and clinical outcomes in patients.ResultsWe found that type I interferon (IFN-I) response was a key differentially regulated pathway between metastatic and non-metastatic cell lines and tumors. The IFN-I response was active in metastatic cancer cells in culture and markedly dampened when these cells formed primary tumors. Interestingly, the opposite was observed in non-metastatic cancer cells and tumors. Consistent with an active IFN-I response in culture, the metastatic cancer cells displayed elevated levels of cytosolic DNA from both mitochondria and ruptured micronuclei with concomitant activation of cGAS-STING signaling. Interestingly, decreased IFN-I-related gene expression in breast cancer biopsies correlated with an unfavourable prognosis in patients.ConclusionOur findings show that IFN-I response is dampened in the tumors with the metastatic ability and lower IFN-I expression predicts poor prognosis in triple-negative and HER2 enriched breast cancer patients. This study highlights the possibility of reactivating the IFN-I response as a potential therapeutic strategy in breast cancer.Graphical abstractEKKkruWu7By9a9jW-1n1_wVideo

Abstract P5-02-03: Combined biomarker analysis for prediction of pathological complete response (pCR) after 12 weeks of pembrolizumab + trastuzumab + pertuzumab in HER2-enriched early breast cancer: Keyriched-1 trial

Abstract Background In unselected HER2+ early breast cancer (EBC), de-escalated chemotherapy-free neoadjuvant therapy (NAT) with dual HER2-blockade induces pCR rates of only 20%-40%. In order to achieve pCR rates by de-escalated therapy comparable to those achieved by chemotherapy-based regimens, patient selection and more effective chemotherapy-free regimens are thus key. KEYRICHED-1 (NCT03988036), a single-arm phase 2 study, is the first trial to investigate chemotherapy-free NAT with dual HER2 blockade and pembrolizumab in HER2-enriched HER2+ EBC. In a translational subproject, we analyzed gene signatures together with tumor cell proliferation and spatiotemporal immune cell profiling to identify predictive factors for pCR. Methods 48 pre- and postmenopausal patients with newly diagnosed HER2 2+ (ISH positive) or 3+ EBC (stage I-III) and HER2-enriched (HER2-E) subtype by PAM50 were included in the study. All patients received 4 cycles of pembrolizumab (200 mg), trastuzumab biosimilar ABP 980 (loading dose (LD) 8 mg/kg bodyweight (BW), maintenance dose (MD) 6 mg/kg BW), and pertuzumab (LD 840 mg/kg BW, MD 420 mg/kg BW) q21d. Primary objective was pCR (centrally confirmed absence of invasive tumor in breast and lymph nodes: ypT0/is, ypN0). NanoString Breast Cancer 360 panel was performed in baseline biopsies (n=42). ≥30% Ki67 decrease, &amp;lt; 500 invasive tumor cells or no evidence of tumor in week 3 biopsies (on treatment) were classified as early response. sTILs were analyzed at baseline (n=42) and week 3 (n=28). Ongoing analyses include whole exome sequencing and multiplexed immunohistochemistry for expression of PD1, PDL1, CD4, CD8, CD68, and CD20 levels in tumor and stroma at baseline and at week 3. Impact of standardized expression of single genes, signatures, and sTILs on pCR was evaluated with univariable and multivariate logistic regression analyses and summarized with odds ratios (OR) and 95% confidence intervals (95%CI). Results 42 patients with BC360 and sTILs data at baseline were included in the analysis. Median age was 55 years (range: 22-83), 11 patients (31%) had node-positive EBC. At baseline, 28 patients had sTIL levels ≥30% and 14 had sTILs &amp;lt; 30%; the corresponding pCR rates were 57.1% (n=16) and 28.6% (n=4, p=0.108). At week 3 (on treatment), 16 patients had sTIL levels ≥30%, 50% (n=8) had a pCR vs 8.3% in those with &amp;lt; 30% sTILs (one patient out of 12, p=0.039). 37 patients had early response, 54.1% of them (n=20) had a pCR vs 0% in early non-responders (n=5, p=0.049). In univariate analysis, IDO1, ERBB2, IFNγ, cytotoxic cells, cytotoxicity, CD8 T-cells, TIGIT, and tumor inflammation signatures were statistically significantly associated with pCR (OR 2.3-3.6); ERBB2, IDO1, IFNγ and CD8 T-cells remained significant after adjusting for hormone receptor (HR) and central HER2 status (OR 2.2-4.3). 70 single genes were predictive for pCR; none of them remained significant after false discovery rate adjustment (25%). In multivariable analysis for baseline markers including signatures, sTILs, HR and central HER2 status, only ERBB2 (OR 8.7, 95%CI 1.9-39.0, p=0.0046) and cytotoxic cells signatures (OR 4.6, 95%CI 1.6-13.5, p=0.0059) were predictive for pCR. Results of whole exome sequencing, and multiplexed immunohistochemistry analysis of immune cell markers will be presented at the Symposium. Conclusions Biomarker analysis in the unique KEYRICHED-1 cohort revealed that early response at week 3, ERBB2 and immune related signatures as well as on-therapy sTIL levels predict pCR after a chemotherapy-free combination of immunotherapy and dual HER2 blockade in HER2-enriched EBC. These results pave the way for validation in larger de-escalation trials investigating short, chemotherapy-free regimens in selected patients with HER2+ EBC. Funding for this research was provided by MSD Sharp &amp; Dohme GmbH. Citation Format: Monika Graeser, Sherko Kuemmel, Oleg Gluz, Friedrich Feuerhake, Valery Volk, Daniel Ulbrich-Gebauer, Claudia Biehl, Mattea Reinisch, Athina Kostara9, Iris Scheffen, Kerstin Luedtke-Heckenkamp, Andreas Hartkopf, Felix Hilpert, Angela Kentsch, Carsten Ziske, Reinhard Depenbusch, Michael Braun, Jens-Uwe Blohmer, Christine zu Eulenburg, Matthias Christgen, Ronald Kates, Stephan Bartels, Hans-Heinrich Kreipe, Enrico Pelz, Peter Schmid, Nadia Harbeck. Combined biomarker analysis for prediction of pathological complete response (pCR) after 12 weeks of pembrolizumab + trastuzumab + pertuzumab in HER2-enriched early breast cancer: Keyriched-1 trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-03.

Abstract P1-13-16: Dissecting the biological activity of different CDK4/6 inhibitors (CDK4/6i) in hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC)

Abstract Background: Palbociclib might be less effective than ribociclib in HR+/HER2- and HER2-enriched BC. This hypothesis is currently being tested in the HARMONIA prospective phase III clinical trial (NCT05207709). Here, we evaluated the downstream biological effects of both drugs using cell lines and patient tumor samples. Methods: Three HR+ BC cell lines (i.e., MCF7, T47D and BT474) were treated at 2 different dose levels of palbociclib or ribociclib (i.e., 100 nM and 500 nM) +/- fulvestrant (1 nM) for 72 hours (h). PAM50 gene signatures were determined on the nCounter, as well as phosphorylation of RB (p-RB) by Western Blot and senescence-associated β-galactosidase activity by FACS. In vitro experiments were performed in triplicates. PAM50 gene signatures were obtained from 49 paired baseline versus week-2 samples and 49 paired baseline versus surgery samples of the CORALLEEN phase II study (Prat, Lancet Oncol. 2020), which treated 49 women with PAM50 Luminal B HR+/HER2- early BC with neoadjuvant ribociclib (600 mg daily) plus endocrine therapy (ET). PAM50 signature scores were also evaluated in publicly available data from 23 paired baseline versus week-2 samples and 16 paired baseline versus surgery samples of the NEOPALANA phase II trial (Ma, Clin Cancer Res. 2017) which treated 50 patients with HR+/HER2- early BC with palbociclib (125 mg daily) plus anastrozole. Changes in PAM50 signatures upon CDK4/6i were determined by paired t-tests and significant analysis of microarray (SAM). Results: Across all cell lines, both palbociclib and ribociclib statistically significantly reduced p-RB at 72h with both doses (i.e., 100 and 500 nM) compared to non-treated cells (p&amp;lt; 0.001). Senescence was also observed at 72h with both doses. Both drugs +/- ET significantly increased the Luminal A signature and decreased Luminal B and proliferation signatures with both doses. However, the HER2-enriched signature was only significantly reduced when both CDK4/6 inhibitors were given at 500 nM. In tumor samples from the CORALLEEN and NEOPALANA phase II studies, a similar change in PAM50 biology was observed with both drugs, namely an increase in Luminal A signature and a decrease in Luminal B and proliferation signatures after 2 weeks of treatment and at surgery. At 2 weeks of treatment, the HER2-enriched signature was significantly decreased in both studies with ribociclib (p&amp;lt; 0.001) and palbociclib (p&amp;lt; 0.001). However, the decrease in the HER2-enriched signature was only observed in surgical samples of patients treated with ribociclib (p&amp;lt; 0.001), but not palbociclib (p=0.194). A difference in sample size could explain this result. Nevertheless, in CORALLEEN, the median number of days between the last dose of ribociclib and surgery was 13.1 days (range: 1-78). In NEOPALANA, the median number of days between the last dose of palbociclib and surgery was 29 days (range: 8-49), except for 8 patients who received additional 10-12 days of palbociclib immediately before surgery (Ma, Clin Cancer Res. 2017). In patients who underwent surgery at 8 days or before, the HER2-enriched signature was significantly decreased for both ribociclib (p&amp;lt; 0.001) and palbociclib (p=0.013). Interestingly, in patients that underwent surgery after &amp;gt;8 days from the last dose, a significant reduction of the HER2-enriched signature was only observed with ribociclib (p&amp;lt; 0.001), but not with palbociclib (p=0.500). Conclusions: Both palbociclib and ribociclib have similar effects on PAM50 biology when given at the same dose. However, in clinical practice, palbociclib is given at a lower dose than ribociclib, and although HER2-enriched signature is significantly decreased in tumors after 2 weeks of CDK4/6i+ET, this effect is only maintained at later time points with ribociclib, indicating a dose-dependent efficacy of CDK4/6i in this biologically aggressive subtype. Citation Format: Natàlia Lorman-Carbó, Olga Martínez-Sáez, Aranzazu Fernandez-Martinez, Patricia Galván, Nuria Chic, Barbara Adamo, Maria Vidal, Montserrat Muñoz, Charles M. Perou, Joaquín Gavilá, Tomás Pascual, Aleix Prat, Fara Brasó-Maristany. Dissecting the biological activity of different CDK4/6 inhibitors (CDK4/6i) in hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-16.

Abstract P1-09-09: Salvage mastectomy is not always necessary for aggressive subtypes of ipsilateral breast cancer recurrence: A single-institution retrospective study

Abstract Introduction: Patients with triple-negative (TN) or HER2-enriched ipsilateral breast cancer recurrence (IBCR) seem to be excluded from a second breast-conserving surgery (BCS) under the assumption that salvage mastectomy would provide better oncological outcomes. Objectives: The objective of this study was to describe the clinical features of these patients, to compare the two surgical alternatives (salvage mastectomy versus second BCS) in terms of oncological results, and to identify independent factors influencing prognosis and surgical treatment. Methods: We retrospectively reviewed all the consecutive patients with histologically confirmed TN or HER2-enriched IBCR. Disease-free survival (DFS), distant disease-free survival (DDFS), overall survival (OS), and breast cancer-specific survival (BCSS) were analyzed and compared between the two groups. Results: Eighty-five patients were affected by TN or HER2-enriched IBCR, with a median age of 60 years (range, 32-87 years). The majority of patients (72.9%) were treated with salvage mastectomy. There was no significant difference in terms of DFS between patients receiving a second BCS or mastectomy (p=0.596). However, patients undergoing a second BCS had significantly better DDFS, OS, and BCSS compared to mastectomy (p=0.009; p=0.002; p=0.001, respectively). Tumor dimension &amp;lt; 16 mm (78.3% versus 38.7%, hazard ratio (HR)=3.602, 95% confidence interval (95% CI)=1.534-8.459, p=0.003) was found to significantly increase the probability of receiving a second BCS and positively affects recurrence and survival outcomes (DFS: HR=8.065, 95% CI=2.320-28.034, p=0.001; DDFS: HR=17.011, 95% CI=3.853-75.099, p=0.001; OS: HR: 13.881, 95% CI=2.730-70.579, p=0.002; BCSS: HR=36.773, 95% CI=4.579-295.322, p=0.001). Second BCS represents an independent protective factor for OS and BCSS (OS: HR=0.246, 95% CI=0.027-0.697, p=0.002; BCSS: HR=0.313, 95% CI=0.092-0.511, p=0.002). Conclusion: Salvage mastectomy is not always necessary and it does not seem to increase survival compared to a second BCS. This reinforces the concept that the prognosis of TN and HER2-enriched BC recurrence is mainly driven by the biology of the disease, rather than by the extent of surgery. In patients with small (&amp;lt; 16 mm) aggressive subtypes of IBCR, a second conservative approach can still be evaluated and offered, presenting acceptable loco-regional control and survival. Citation Format: Damiano Gentile, Andrea Sagona, Erika Barbieri, Simone Di Maria Grimaldi, Ruggero Spoto, Davide Franceschini, Stefano Vaccari, Valeriano Vinci, Ersilia Biondi, Lorenzo Scardina, Corrado Tinterri. Salvage mastectomy is not always necessary for aggressive subtypes of ipsilateral breast cancer recurrence: A single-institution retrospective study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-09-09.

Predictive and Prognostic Role of Tumor-Infiltrating Lymphocytes in Patients with Advanced Breast Cancer Treated with Primary Systemic Therapy.

Tumor-infiltrating lymphocytes (TILs) are gaining recognition as an important immunological biomarker with therapeutic potential in breast cancer. In this cohort study conducted on patients with advanced breast cancer treated with primary systemic therapy (PST), the TILs concentration was correlated with response to PST and survival outcomes. Patients with primary breast cancer treated with PST between 2016 and 2020 were included in this study, approved by IEC, and registered on ClinicalTrials.gov (NCT05250336). Tumor core biopsies obtained prior to starting treatment from 489 patients were assessed for the proportion of stromal TILs by standardized method and categorized into low (0-10% immune cells), intermediate (11-59%), and high (≥ 60%) TILs. TIL concentration and complete pathological response (pCR), disease-free survival (DFS), and overall survival (OS) were correlated. Of the 489 patients, 372 matched the eligibility criteria for assessment of TILs and made the final study cohort. Among these, 135 were luminal, 129 HER2-enriched, and 108 triple-negative breast cancers (TNBC). Proportions of patients with high TILs were greater in TNBC (15.7%) and HER2-enriched (9.3%), compared to luminal cancers (4.4%). High TIL concentration was correlated with higher pCR in all subtypes. A pCR was achieved in 33.3, 50, and 52.9% of high TIL patients in luminal, HER2-enriched, and TNBC subtypes, respectively (p < 0.05). High TILs were linked to longer DFS and OS in TNBC and HER2-enriched breast cancers. In this first study of its kind from a low- and middle-income country, high TILs concentration was found to be a predictor of response to PST across all breast cancer subtypes. TILs concentration was found predictive of better DFS and OS in TNBC and HER2-enriched cancers. Prognostic role of TILs in luminal cancers was not so apparent.

Clinical verification of the relationship between serum lipid metabolism and immune activity in breast cancer patients treated with neoadjuvant chemotherapy

BackgroundLipid metabolism has been recently reported to affect the prognosis and tumor immune activity in cancer patients. However, the effect of lipid metabolism on chemosensitivity in patients with breast cancer treated with neoadjuvant chemotherapy (NAC) remains unclear.MethodsWe examined 327 patients with breast cancer who were treated with NAC followed by curative surgery. The correlations between the serum levels of total cholesterol (TC) and triglyceride (TG) and the clinicopathological features, including the efficacy of NAC, neutrophil-to-lymphocyte ratio (NLR), and absolute lymphocyte count (ALC), were evaluated retrospectively.ResultsSerum TG levels were increased after NAC in all the subtypes, and the rate of change was the highest, especially in triple-negative breast cancer (TNBC) (21.0% → 48.1%). In addition, only TNBC patients with an objective response (OR) had significantly higher TG levels after NAC than those without (P = 0.049). Patients with a high ALC before NAC had significantly higher TG levels after NAC than patients with all breast cancer (P = 0.001), HER2-enriched breast cancer (P = 0.021), and TNBC (P = 0.008). Patients with a low NLR before NAC had significantly higher TG levels after NAC only among patients with TNBC (P = 0.025). In patients with human epidermal growth factor receptor 2-enriched breast cancer, the group with normal TC levels before NAC had significantly better OS than those with high TC levels (P = 0.013, log-rank test), and in patients with TNBC, the group with high TC levels after NAC had significantly better OS than those with normal TC levels (P = 0.014, log-rank test).ConclusionsGood systemic immune activity and chemosensitivity may be associated with lipid metabolism regulated by NAC in TNBC patients.

Multiparametric MRI Features of Breast Cancer Molecular Subtypes.

Background and Objectives: Breast cancer (BC) molecular subtypes have unique incidence, survival and response to therapy. There are five BC subtypes described by immunohistochemistry: luminal A, luminal B HER2 positive and HER2 negative, triple negative (TNBC) and HER2-enriched. Multiparametric breast MRI (magnetic resonance imaging) provides morphological and functional characteristics of breast tumours and is nowadays recommended in the preoperative setting. Aim: To evaluate the multiparametric MRI features (T2-WI, ADC values and DCE) of breast tumours along with breast density and background parenchymal enhancement (BPE) features among different BC molecular subtypes. Materials and Methods: This was a retrospective study which included 344 patients. All underwent multiparametric breast MRI (T2WI, ADC and DCE sequences) and features were extracted according to the latest BIRADS lexicon. The inter-reader agreement was assessed using the intraclass coefficient (ICC) between the ROI of ADC obtained from the two breast imagers (experienced and moderately experienced). Results: The study population was divided as follows: 89 (26%) with luminal A, 39 (11.5%) luminal B HER2 positive, 168 (48.5%) luminal B HER2 negative, 41 (12%) triple negative (TNBC) and 7 (2%) with HER2 enriched. Luminal A tumours were associated with special histology type, smallest tumour size and persistent kinetic curve (all p-values < 0.05). Luminal B HER2 negative tumours were associated with lowest ADC value (0.77 × 10−3 mm2/s2), which predicts the BC molecular subtype with an accuracy of 0.583. TNBC were associated with asymmetric and moderate/marked BPE, round/oval masses with circumscribed margins and rim enhancement (all p-values < 0.05). HER2 enriched BC were associated with the largest tumour size (mean 37.28 mm, p-value = 0.02). Conclusions: BC molecular subtypes can be associated with T2WI, ADC and DCE MRI features. ADC can help predict the luminal B HER2 negative cases.

The prognostic significance of further axillary dissection for sentinel lymph node micrometastases in female breast cancer: A competing risk analysis using the SEER database.

Sentinel lymph node (SLN) biopsy has been widely recognized as an excellent surgical and staging procedure for early-stage breast cancer, and its development has greatly improved the detection of micrometastases. However, the axillary treatment of micrometastasis has been the subject of much debate. We identified 427,131 women diagnosed with breast cancer from 2010 to 2018 in the Surveillance, Epidemiology, and End Results (SEER) database. Patients whose nodal status was micrometastases (pTxN1miM0) were classified into two groups: the SLNB only group and SLNB with complete ALND group, and we used these classifications to carry out propensity-score matching (PSM) analysis. The primary and secondary endpoints were OS and BCSS, respectively. We then implemented the Kaplan-Meier method and Cox proportional hazard model and used Fine and Gray competitive risk regression to identify factors associated with the risk of all-cause mortality. After the PSM, 1,833 pairs were included in total. The SLNB with complete ALND showed no significant difference in OS (HR=1.04, 95% CI: 0.84-1.28, P=0.73) or BCSS (HR= 1.03, 95% CI: 0.79-1.35, P=0.82) compared to the SLNB only group, and axillary treatment was not associated with breast cancer-specific death (BCSD) (HR=1.13, 95% CI: 0.86-1.48, P=0.400) or other cause-specific death (OCSD) (HR=0.98, 95% CI:0.70-1.38, P=0.920). There was no statistically significant difference in the cumulative incidence of BCSD (Grey's test, P=0.819) or OCSD (Grey's test, P=0.788) for between the two groups either. For different molecular subtypes, patients in the SLNB only group showed no statistically significant differences from those in the SLNB with complete ALND group with Luminal A (HR=1.00, 95% CI:0.76-1.32, P=0.98) or Luminal B (HR=0.82, 95% CI:0.42-1.62, P=0.55) but similar OS to HER2-enriched (HR=1.58, 95% CI:0.81-3.07, P=0.19) or triple negative breast cancers (HR=1.18, 95% CI:0.76-1.81, P=0.46). Our results suggest that in early breast cancer patients with micrometastasis, complete ALND does not seem to be required and that SLNB suffices to control locoregional and distant disease, with no significant adverse effects on survival compared to complete ALND.

Mass spectroscopy-based proteomics and metabolomics analysis of triple-positive breast cancer cells treated with tamoxifen and/or trastuzumab.

HER2-enriched breast cancer with high levels of hormone receptor expression, known as "triple positive" breast cancer, may represent a new entity with a relatively favourable prognosis against which the combination of chemotherapy, HER-2 inhibition, and endocrine treatment may be considered overtreatment. We explored the effect of the anticancer drugs tamoxifen and trastuzumab, both separately and in combination, on the integrated proteomic and metabolic profile of "triple positive" breast cancer cells (BT-474). We employed ultra-high-performance liquid chromatography-quadrupole time of flight mass spectrometry using a Bruker timsTOF to investigate changes in BT-474 cell line treated with either tamoxifen, trastuzumab or a combination. Differentially abundant metabolites were identified using the Bruker Human Metabolome Database metabolite library and proteins using the Uniprot proteome for Homo sapiens using MetaboScape and MaxQuant, respectively, for identification and quantitation. A total of 77 proteins and 85 metabolites were found to significantly differ in abundance in BT-474 treated cells with tamoxifen 5μM/and or trastuzumab 2.5μM. Findings suggest that by targeting important cellular signalling pathways which regulate cell growth, apoptosis, proliferation, and chemoresistance, these medicines have a considerable anti-growth effect in BT-474 cells. Pathways enriched for dysregulation include RNA splicing, neutrophil degranulation and activation, cellular redox homeostasis, mitochondrial transmembrane transport, ferroptosis and necroptosis, ABC transporters and central carbon metabolism. Our findings in protein and metabolite level research revealed that anti-cancer drug therapy had a significant impact on the key signalling pathways and molecular processes in triple positive BT-474 cell lines.

Higher Expressions of SHH and AR Are Associated with a Positive Receptor Status and Have Impact on Survival in a Cohort of Croatian Breast Cancer Patients.

Breast cancers (BC) are usually classified into four molecular subtypes according to the expression of estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER2) receptors and proliferation marker Ki-67. Despite available anti-hormonal therapies and due to the inherent propensity of some subtypes to develop metastasis, there is a permanent need to discover new prognostic and predictive biomarkers, as well as therapeutic targets for BC. In this study, we used immunohistochemical staining to determine the expression of androgen receptor (AR) and sonic hedgehog protein (SHH), the main ligand of the Hedgehog-GLI (HH-GLI) signaling pathway, in 185 archival primary BC tissue samples and correlated it with clinicopathological characteristics, molecular subtypes, receptors statuses, and survival in a cohort of Croatian BC patients. Results showed that higher SHH and AR expressions were associated with positive receptor status, but increased SHH expression had a negative impact on survival in receptor-negative BCs. On the contrary, higher AR expression was mostly protective. However, multivariate analysis showed that only higher AR expression could be considered as an independent prognostic biomarker for poorer overall survival in triple-negative breast cancer patients (TNBC) (HR 10.9, 95% CI 1.43–83.67; p = 0.021), what could be Croatian population-related. SHH could be a potential target for treating TNBCs and HER2-enriched BCs, in cases where HH-GLI signaling is canonical (SHH-dependent).

Quantitative Analysis of Contrast-enhanced Mammography for Risk Stratification of Benign Versus Malignant Disease and Molecular Subtype.

To assess quantitative enhancement of benign, high-risk, and malignant lesions and differences in molecular subtype and grade of malignant lesions on contrast-enhanced mammography (CEM). This IRB-approved retrospective study included women who underwent CEM for diagnostic work-up of a breast lesion between 2014 and 2020. Inclusion criteria were women who had diagnostic work-up with CEM and had BI-RADS 1 or 2 with one year follow-up, BI-RADS 3 with tissue diagnosis or stability for 2 years, or BI-RADS 4 or 5 with tissue diagnosis. An enhancement ratio was calculated for all lesions. This was obtained by drawing a region of interest within the lesion and a second region of interest in the nonenhancing background tissue using a program developed with MATLAB. Descriptive statistics were evaluated using chi-squared tests, Fisher exact tests, and analysis of variance. A logistic regression model was used to predict cancer outcome using the enhancement ratio. Statistical significance was defined as P < 0.05. There were 332 lesions in 210 women that met study criteria. Of the 332 lesions, 50.9% (169/332) were malignant, 5.7% (19/332) were high-risk, and 43.4% (144/332) were benign. Enhancement intensity of malignant lesions was higher than benign lesions. Odds ratio for quantitative enhancement of malignant lesions was 30.15 (P < 0.0001). Enhancement ratio above 1.49 had an 84.0% sensitivity and 84.0% specificity for malignancy. HER2-enriched breast cancers had significantly higher mean enhancement ratios (P = 0.0062). Quantitative enhancement on CEM demonstrated that malignant breast lesions had higher mean enhancement intensity than benign lesions.

Abstract 92: tGLI1 and IL-6/IL-6R/GP130/STAT3 pathways cooperate to promote breast cancer stem cells and are co-activated in HER2-enriched and triple-negative breast cancers

Abstract Breast cancer is the most frequently diagnosed cancer and second leading cause of cancer-related deaths in women; 30% of primary breast cancer cases will develop distant metastases. The majority (90%) of breast cancer-related deaths are due to distant metastases. HER2-enriched breast cancer and triple-negative breast cancer (TNBC) have the highest propensity to metastasize and have limited to no treatment options. Metastatic HER2-enriched breast cancer and TNBC patients commonly develop therapeutic resistance and, therefore, lack efficacious therapies. Due to dismal prognoses in metastatic HER2-enriched breast cancer and TNBC patients, there remains an urgent need to identify novel molecular pathways for actionable therapeutic targets. Truncated glioma-associated oncogene homolog 1 (tGLI1), an alternatively spliced variant of GLI1, is a gain-of-function tumor-specific terminal effector in the Hedgehog pathway. tGLI1 promotes breast cancer growth and circulating tumor cells to the brain. More recently, our lab has reported tGLI1 physically interacts with Signal Transducer and Activator of Transcription 3 (STAT3); tGLI1 and STAT3 are co-overexpressed in node-positive HER2-enriched and triple-negative breast carcinomas. The Interleukin-6 (IL-6) signaling axis is a primary regulator of STAT3 and is frequently dysregulated and hyperactivated in multiple cancers, including breast cancer. IL-6 functions through a hexameric receptor complex comprised of IL-6 receptor α (IL-6R) and glycoprotein 130 (GP130) to recruit Janus tyrosine kinases (JAK). In turn, JAKs phosphorylate STAT3 resulting in homodimerization, nuclear translocation, and transcriptional activity. Independently, tGLI1 and IL-6/IL-6R/GP130/STAT3 pathways have been previously investigated as mediators for breast cancer metastases; however, whether they cooperate to promote breast cancer progression remains unknown. Herein, we report tGLI1 and IL-6/IL-6R/GP130/STAT3 pathways to be frequently co-expressed and co-activated in HER2-enriched breast cancer and TNBC tumors and that the co-activation is associated with a shortened metastasis-free survival (MFS). Additionally, when stratified by metastatic site, breast tumors with high tGLI1 and IL-6/IL-6R/GP130/STAT3 co-activation are associated with a worse bone, brain, and lung MFS compared to tumors with low co-activation. Ectopic co-overexpression of tGLI1 and GP130 upregulates mRNA expression of stemness genes, Nanog, Sox2, and Oct4, leading to the subsequent promotion of the breast cancer stem cell (CSC) subpopulation in vitro. Taken together, our findings revealed a novel crosstalk between tGLI1 and the IL-6/IL-6R/GP130/STAT3 signaling axis to enhance the CSC subpopulation, suggesting the importance of the crosstalk in mediating metastasis of HER2-enriched breast cancer and TNBC. Citation Format: Sara G. Manore, Daniel L. Doheny, Dongqin Zhu, Grace L. Wong, Angelina T. Regua, Austin Arrigo, Calvin J. Wagner, Mariana Najjar, Hui-Wen Lo. tGLI1 and IL-6/IL-6R/GP130/STAT3 pathways cooperate to promote breast cancer stem cells and are co-activated in HER2-enriched and triple-negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 92.

Abstract 1039: TrkA and JAK2-STAT3 pathway crosstalk promotes breast cancer stem cells in HER2-enriched and triple-negative breast cancers

Abstract Breast cancer is the most commonly diagnosed cancer in American women and accounts for ~15% of cancer-related deaths. Despite the current standard of care, metastatic HER2-enriched breast cancer and triple-negative breast cancer remain difficult to treat due to poor response to treatment, lack of actionable targets, or eventual acquired resistance. The high mortality rate of metastatic HER2-enriched breast cancers and triple-negative breast cancers highlights the need for novel actionable targets for improved response to therapeutic intervention. Through datamining of publicly available breast cancer patient datasets, we recently found that Tropomyosin receptor kinase A (TrkA) and Janus kinase 2 (JAK2)-STAT3 pathways are co-activated and co-enriched in HER2-enriched breast cancers and triple-negative breast cancers (Cancers 10:2340-2360, 2021). We also found that TrkA, a receptor tyrosine kinase, directly interacts with and phosphorylates STAT3 on Y705 residue, resulting in STAT3 activation, nuclear translocation, and increased transcription of STAT3 target genes SOX2 and c-MYC, which are associated with breast cancer stem cell formation as well as tumor recurrence and metastasis. In this study, we aimed to further characterize the effects of the TrkA-JAK2/STAT3 pathway crosstalk on breast cancer stem cells. Our data showed that overexpression of TrkA enhances mammosphere formation and ALDH activity of breast cancer cells, which are further enhanced upon co-overexpression of STAT3. Our study further revealed that TrkA and JAK2 inhibitors synergize to reduce breast cancer stemness since co-inhibition of TrkA and JAK2 significantly reduces the CD44high/CD24low cell subpopulation, mammosphere formation, and ALDH activity to a greater extent compared to vehicle or monotherapies. Western blot analysis of breast cancer cells treated with TrkA and/or JAK2 inhibitors showed that co-inhibition of these two kinases significantly reduces levels of p-STAT3 (Y705), as well as stemness markers SOX2, MYC, and CD44. Taken together, these findings suggest that TrkA cooperates with the JAK2-STAT3 signaling pathway to promote breast cancer stem cells through increasing expression of SOX2, c-MYC, and CD44, and that co-inhibition of TrkA and JAK2 significantly suppressed breast cancer stemness, suggesting its future utility as a promising new treatment modality for patients with HER2-enriched breast cancers and triple-negative breast cancers. Citation Format: Angelina T. Regua, Dongqin Zhu, Daniel L. Doheny, Grace L. Wong, Sara G. Manore, Calvin J. Wagner, Austin Arrigo, Mariana Najjar, Hui-Wen Lo. TrkA and JAK2-STAT3 pathway crosstalk promotes breast cancer stem cells in HER2-enriched and triple-negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1039.